The overall goal of this application is to expand our studies on a novel approach for assessing Type 1 diabetes risk in humans. This approach relies on a minimally invasive procedure such as the analysis of autoreactive CD4 T cells directly in the blood using FACS and specific ligands for cognate TCR/CD4 (soluble, dimeric MHC ll-peptide, DEF). Quantifying and phenotyping autoreactive CD4 T cells in mice allowed us not only to predict the onset of diabetes but also to gain insights into the pathogenesis of T1D. We found that disease progression is marked by an imbalance between regulatory CD4+CD25+ and activated CD4+CD69+ (autoreactive) T cells in pancreas, and that such event is mirrored in the phenotype of autoreactive CD4 T cells circulating in the blood. Our studies also revealed the "hit-and-run" nature of the autoimmune attack on pancreas by recruitment of activated (CD69+) T cells from the blood. Several such "hits" damage enough number of beta-cells so that the insulin production decreases and over the time leads to the onset. Our studies in humans also showed the potential of this approach for assessing T1D risk. We found that autoreactive CD4 T cells specific for GAD65 autoantigen are commonly present in blood of those expressing diabetes-susceptible HLA-DR*0401 molecules. The risk for diabetes can be assessed upon the imbalance between CD4+CD25+ and CD4+CD69+ T cells specific for GAD65 T cells. Like we found in mice, the autoreactive T cell imbalance followed by the disappearance of these T cells from the blood (the "hit") associated in some cases with the clinical onset of T1D. [unreadable] [unreadable] In this application, we pursue 2 specific aims: In aim 1 we will test the hypothesis that tracing autoreactive CD4 T cells in peripheral blood is a specific marker of the autoimmune process that leads to human T1 D. In aim 2 we will test the hypothesis that the autoimmune attack on pancreas is a hit-and-run process mediated by the recruitment of activated T cells from the blood. Relatives of patients with T1D and controls will undergo physiological examination for insulin release to correlate changes in the specific CD4 T cells in blood with pancreatic beta-cell function. This study is expected to increase our understanding of T1D pathogenesis and provide new methods for assessing T1D risk. [unreadable] [unreadable] [unreadable]